IBL-AMERICA ELISA TROUBLESHOOTING GUIDEPROBLEMPOSSIBLE CAUSESOLUTION/REMEDY

Reagents added/prepared
incorrectly

Check protocol, ensure that the reagents were addedin the proper order and prepared to correct dilution.

Reagents not equilibrated to room
temperature
Incorrect storage ofreagents/Expired Reagents
Very low incubation temperature oragitation.

Weak or No Signal
Wells scratched with pipette orwashing tips.

Use caution when dispensing and aspirating into andout of wells. Automated plate washers may need to becalibrated so tips don't touch the bottom of the wells.

Plate washing to forceful

Check and ensure correct pressure in automatic plate
washer. Pipette wash buffer gently if manuallywashing the plate.

Plate read at incorrect wavelength

Double check product protocol to ensure plate reader
is set to the correct wavelength.

Incompatible sample type

Insufficient washingHigh Background
Across the Plate.
Standard Curve isSaturated.
Too Much Signal

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Per specific product protocol ensure appropriate
reagents equilibrate to room temperature prior tostarting the assay.
Double check the expiration dates and storageconditions in product protocol and/or component
labels.
Control your room (lab) temperature to bring it within
the recommended range (Refer to the Package
Insert). Set your plate shaker to 600rpm.

Detection may be reduced or absent in untested
sample types. Include a sample that the assay is
known to detect as a positive control.
Wash wells as per the product protocol. As a generalrule, increasing the duration of the soak step may help
(Add 30 seconds to the soak time). In addition, at theend of each washing step, invert the plate onabsorbent tissue and tap forcefully if necessary toremove any residual fluid.

Contaminated wash buffer

Prepare a fresh solution of wash buffer.

Wrong conjugate dilution used.

Dilute the conjugate at the recommended dilution.

The assay was incubated for too
long in one or all steps.

Strictly follow the assay incubation time in all steps
according to the procedure.

Substrate incubation not protectedfrom light

Substrate incubations are commonly performed in thedark. Review the product protocol and follow
recommendation of the manufacturer.

The substrate is contaminated (notfresh).

Check the color of the substrate - it should becolorless.

High incubation temperature.

Control your room (lab) temperature to bring it within
the recommended range (Refer to the Package
Insert).

Dirty plate/well bottom

Clean the bottom of the plate.

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Positive Signal (highbackground) in
Negative Controls orStandard Wells.

Contaminated Pipette tips.

Use clean pipette tips and change tips for every
sample and/or standard.

Cross contamination has occurred
between wells.

Carefully wash wells. The wash step is very critical.

Incorrect standard dilutions prepared

Review protocol and double check calculations toensure proper dilution of standards/controls

Incorrect standard curve dilutions
prepared

Confirm dilutions are made correctly and avoid crosscontamination of standards by utilizing new pipettetips with each standard preparation.

Standard improperly reconstituted

Briefly spin vial prior to opening. Inspect vial for
undissolved material after reconstitution.

Poor Standard Curvein ELISA

Possible degradation of standard
material
Curve doesn't fit data

Pipetting problem.

Ensure storage and handling of the standard wasproperly followed as recommended in the productprotocolTry plotting standard data using a different scale.
e.g. log-log, 4 Parameter logistic, 5 parameter logistic
curve fits.
When using:Single Channel Pipette: Check tips for bubbles
between replicates.
Multichannel Pipette: Calibrate the pipette. Check tips
for bubbles before dispensing.

Bubbles in wells

High CV across theplate (high variationin samples /standards).

Sample Readings
are out of the assay
dynamic range.

2Edge Effects

Insufficient washing

Ensure no bubbles are present prior to reading theplate.
Ensure the plate and all reagents are at room
temperature.
Wash wells as per the product protocol. As a generalrule, increasing the duration of the soak step may help
(Add 30 seconds to the soak time). In addition, at theend of each washing step, invert the plate on
absorbent tissue and tap forcefully if necessary toremove any residual fluid.

Non-Uniform Washing.

All the wells should be uniformly washed. Check yourwashing system and/or method.

Inconsistent sample storage

Ensure sample storage conditions are maintained per
guidelines outlined in the assay protocol.

Non-homogeneous reagents,
samples, or standards

Ensure all reagents are mixed thoroughly. Vortex
samples/standards to ensure homogeneity before
pipetting.

Sample contains undetectable
analysis level, i.e. below assay limitof detection (LOD).
Sample contains high analyte level:
i.e. above assay highest standardpoint.

If samples fall below the LOD, contact us: info@iblamerica.com
Samples should be diluted and re-assayed.

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Pipetting Technique Tips for ELISA
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Use only pipettes within the range suggested by the manufacturer.
Make sure disposable pipette tip is fit tightly onto the pipette
Check that there are no air bubbles while pipetting reagents/samples/standards/controls.
Reduce splashing of liquid by pipetting into the sides of the microplate wells.
Use a new pipette tip between pipetting of standards, controls, and samples

Online Software for Curve Analysis and Calculation of Results*These programs are not affiliated with IBL-America and should be used at the discretion of the enduser.
1. https://www.myassays.com/
2. http://www.elisaanalysis.com/app

If you have any additional questions please contact us directly.

8201 Central Ave NE, Suite P
Minneapolis, MN 55432, USA
info@ibl-america.com(888) 523-1246
https://www.ibl-america.com/

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